Apicomplexan parasites cause many diseases in humans, including malaria and toxoplasmosis. They do this by invading, replicating and exiting human cells. In recent years, much has been learned about the molecules that carry out these processes but how their actions are coordinated and how signals are received to initiate each process have remained largely mysterious. From studies in other organisms, it is known that phosphorylation of proteins is one of the most important means by which their action is regulated. Recently, mass spectrometric techniques have emerged that allow the large-scale identification of the nearly entire set of such modifications; this dataset is known as the phosphoproteome. The goal of our proposed work is to determine the phosphoproteomic changes that occur in the acute stage of Toxoplasma parasites, the so-called tachyzoites, under intracellular vs. egressing conditions. To enable us to determine which changes are key to these processes, we will use specific drugs that disrupt calcium-dependent events that are known to be key to invasion and egress. We will also make strategic use of existing mutant lines of Toxoplasma that are defective in signaling related to invasion/egress. Finally, we will take advantage of our expertise in mass spectrometry to innovate new methods for phosphoproteomic analyses of samples where the amount available is too small for conventional analyses. PUBLIC HEALTH RELEVANCE: The work described here is relevant to the control of important parasitic diseases such as toxoplasmosis and malaria. Specifically, it will increase our understanding of the mechanisms used by these parasites to enter and exit cells. It also has the potential to improve our ability to study the molecular changes that occur in a variety of conditions, including other infections and cancer.